Method of increasing bioavailability and/or prolonging ophthalmic action of a drug

ABSTRACT

The present invention relates to a method of increasing the bioavailability and/or prolonging ophthalmic action of a drug, the method comprising instilling into the eye an aqueous suspension comprising reversible clusters of drug loaded nano-resin particles, said clusters having a D50 value of at least 2 micrometer and said drug loaded nano-resin particles have a particle size distribution characterized in that the D90 value is 70 nanometer to 900 nanometer. The present invention further relates to an aqueous suspension comprising reversible clusters of drug loaded nano-resin particles, said clusters have a D50 value of at least 2 micrometers and said drug loaded nano-resin particles have a particle size distribution characterized in that the D90 value is 70 nanometers to 900 nanometers.

FIELD OF INVENTION

The present invention relates to a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, themethod comprising instilling into the eye an aqueous suspensioncomprising (a) reversible clusters of drug loaded nano-resin particles,said clusters having a D₅₀ value of at least 2 micrometer and said drugloaded nano-resin particles have a particle size distributioncharacterized in that the D₉₀ value is 70 nanometer to 900 nanometer,and (b) a suspending agent.

The present invention also relates to an aqueous suspension comprising(a) reversible clusters of drug loaded nano-resin particles, saidclusters have a D₅₀ value of at least 2 micrometers and said drug loadednano-resin particles have a particle size distribution characterized inthat the D₉₀ value is 70 nanometers to 900 nanometers, and (b) asuspending agent.

BACKGROUND OF THE INVENTION

Ophthalmic drug delivery is one of the most challenging endeavors facingthe pharmaceutical scientist. The eye is a unique organ, bothanatomically and physiologically, containing several widely variedstructures with independent physiological functions. The complexity ofthe eye provides unique challenges to drug delivery strategies.Typically, the ocular bioavailability of drugs applied topically aseye-drops is very poor. The absorption of drugs in the eye is severelylimited by some protective mechanisms that ensure the proper functioningof the eye, and by other concomitant factors, for example: nasolacrimaldrainage of the instilled solutions; lacrimation and tear turnover; lowcorneal contact time; metabolism; tear evaporation; non-productiveabsorption/adsorption; limited corneal area and poor cornealpermeability; and binding by the lacrimal proteins. These factors have ahuge effect on ocular drug absorption and disposition and lead to lowocular drug bioavailability. Thus, developing an ocular drug deliverysystem that provides optimum drug bioavailability is a challenge. It isimportant to consider a number of factors, including effective cornealapplication to promote good corneal penetration, prolonged contact timewith the corneal epithelium and the use of a formulation withappropriate rheological properties that is non-irritable to the eye.This challenge becomes all the more difficult in case of diseases thatare associated with tissues at posterior segment of the eye such asdiabetic retinopathy, glaucomatous optic neuropathy, age-related maculardegeneration etc., which are very difficult to treat. Methods used forocular drug delivery for the front of the eye or anterior segment differsignificantly and pose considerably less risk than subcutaneous orposterior segment eye therapy. Methods available for posterior segmentdrug delivery are complex, for instance injections in the desiredposterior tissue, sustained-release implants, iontophoretic drugdelivery etc. and these can be associated with greater risk ofinfection, internal ocular bleeding and retinal damage. The conventionalophthalmic solutions, suspensions and ointment dosage forms are nolonger sufficient for combating some present virulent diseases of theanterior segment as well as diseases affecting the posterior segment ofthe eye. Moreover, the conventional formulations have anotherdisadvantage that they necessitate frequent administrations, sometimesfour to five times a day in order to provide desired therapeutic effect,which leads to patient non-compliance.

Few advance ocular delivery systems have been commercialized recently orare under development, with an aim at enhancing the drug bioavailabilityeither by providing sustained delivery to the eye or by facilitatingtranscorneal penetration. Advances in recent years in topical oculardrug delivery have ranged from iontophoretic drug delivery, in situgelling systems, dendrimers, penetration enhancers, lipid emulsions,ocular inserts, and site-specific drug delivery systems. Nonetheless,very few drug delivery systems have successfully appeared on the market:currently, about 95% of the products are delivered via the traditionaleye-drop bottle. There is a continuing need for developing efficientophthalmic drug delivery systems/formulations which overcomes theaforesaid problems. There is a need for efficient ophthalmic drugdelivery systems which upon ocular administration, leads to increase inocular bioavailability of the drug both in the anterior as well asposterior segment of the eye, prolonging the ophthalmic action of thedrug, and minimizing the irritation or other discomfort associated withocular application.

The present invention fulfills this need and provides a novel ophthalmicdrug delivery system in the form a novel aqueous suspension comprisingnano-resin particles. The present invention provides a method ofincreasing the bioavailability and/or prolonging the ophthalmic actionof a drug, by instilling the novel aqueous suspension of the presentinvention into the eyes. The ability of the present invention to deliverthe drug in the posterior segment of the eye make it suitable fortreating diseases of the posterior segment, which are difficult totreat.

SUMMARY OF THE INVENTION

The present invention provides a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, themethod comprising instilling into the eye an aqueous suspensioncomprising (a) reversible clusters of drug loaded nano-resin particles,said clusters having a D₅₀ value of at least 2 micrometer and said drugloaded nano-resin particles have a particle size distributioncharacterized in that the D₉₀ value is 70 nanometer to 900 nanometer,and (b) a suspending agent.

The present invention further provides an aqueous suspension comprising(a) reversible clusters of drug loaded nano-resin particles, saidclusters have a D₅₀ value of at least 2 micrometers and said drug loadednano-resin particles have a particle size distribution characterized inthat the D₉₀ value is 70 nanometers to 900 nanometers, and (b) asuspending agent.

The present invention also provides a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, themethod comprising providing an aqueous suspension comprising (a)reversible clusters of drug loaded nano-resin particles, said clustershaving a D₅₀ value of at least 2 micrometers and said drug loadednano-resin particles having a particle size distribution characterizedin that the D₉₀ value is 70 nanometers to 900 nanometers, and (b) asuspending agent

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustrative histogram for the resin particles having meanparticle size of 5 microns used in the preparation of suspension ofcomparative example 1.

FIGS. 2-7 are illustrative histograms for particle size distribution ofthe drug loaded resin particles in the suspension prepared according toComparative Example 1, upon application of shear by subjecting tosonication and measuring particle size distribution after one, two,three, four and five minutes, respectively. The histograms reveal thatthere is no change in the particle size upon application of shear.

FIG. 8 is an illustrative histogram for the nano-resin particles used inthe preparation of aqueous suspension of the present invention.

FIG. 9 is the histogram showing particle size distribution of reversibleclusters/agglomerates of drug-loaded nano-resin particles preparedaccording to Example 2 (no shear applied). There are micron sizeclusters along with few nano-sized resin particles having D₉₀ less than900 nm.

FIG. 10-14 shows histogram showing reversible clusters of drug-loadednano-resin particles being declustered into individual drug-loadednano-resin particles when subjected to application of shear as perExample 7 of the specification.

FIGS. 15-17 provides bar graph representation of values of C_(max) andAUC_(0-t) in different ocular tissues, obtained following ocularadministration of aqueous suspension of the present invention (Example2(B)) and reference formulation (available under the brandname:Alphagan® P), according to study described in Example 11 of the presentinvention.

FIG. 18 provide bar graph representation of reduction in intraocularpressure (mm of Hg) upon instillation of aqueous suspension of thepresent invention i.e. Example 2(A); and Alphagan®P at different timepoints according to the study described in Example 8.

FIG. 19 provide bar graph representation of reduction in intraocularpressure (mm of Hg) upon instillation of two test formulations i.e.aqueous suspension of Example 2(A); aqueous suspension of Example 2(B);and Alphagan®P at different time points according to the study describedin Example 9.

DETAILED DESCRIPTION OF THE INVENTION

‘Reversible Clusters’ of drug loaded nano-resin particles, according tothe present invention means that the individual drug loaded nanoresinparticles form aggregates or agglomerates having mean size of about 2micrometers or greater which upon application of shear deagglomerate ordecluster into individual drug loaded nano-resin particles.

Inventors have found that such reversible clusters decluster uponapplication of shear such as that resulting from the blinking of theeye.

Qualitatively, the declustering can be observed by microscopy(Morphology G3S-ID Instrument, Make: Malvern) by observing sheared (bysmearing) and unsheared samples onto the glass slide.

As referred to further herein, the D₅₀ of the clusters is obtained fromthe particle size distribution obtained before the application of shear.The particle size distribution may be determined in a suitable particlesize analyser such as the Malvern Mastersizer. The particle sizeanalyser's sonication means are not used but the sample is onlysubjected to stirring by a mechanical stirrer.

The suspension of clusters is placed in a sonication bath and issubjected to shear using sonication frequency of about 33±3 kHz for 5seconds, and a sample withdrawn to measure the particle sizedistribution. Following intervals of 1 minute each the process isrepeated 5 times. (See Example 7, FIGS. 9-14)

Nanoresin particles or drug-loaded nanoresin particles according to thepresent invention means individual ion exchange resin particles and notclusters or agglomerates of the individual particles which individualparticles have a particle size distribution characterized in that theD₉₀ value is in the range of about 70 nanometer to 900 nanometers. Theparticle size distribution of the nanoresin particles may be consideredas the particle size distribution obtained after the suspension has beensubjected to 5 pulses of frequency of 33±3 KHz with intervals of 1minute each, as described above.

The individual drug loaded nano-resin particles have a particle sizedistribution such that value of D₉₀ is 70 nanometer to 900 nanometer (nmor nms), preferably 200 nms to 700 nms. Further wherein, suchdrug-loaded nanoresin particles have a mean particle size (D₅₀) of lessthan 500 nms.

Preferably, the D₅₀ value is 50 nanometers to 350 nanometers. Theparticle size distribution of the nanoresin may be further characterizedin that the D₁₀ is less than 300 nms, preferably 10 nms to 250 nms.

According to the present invention, the reversible clusters have aparticle size distribution such than mean size is at least 2 micrometer(μm or microns). In preferred embodiments, the particle sizedistribution is such than the D₉₀ is less than 80 micrometers,preferably less than 50 micrometers, most preferably less than 30micrometers. The particle size distribution may be further characterizedin than the Ds) is less than 40 micrometers, preferably less than 20micrometers, more preferably less than 10 micrometers. The reversibleclusters, upon application of shear deagglomerate or deaggregate to formdrug loaded nanoresin particles.

The present invention provides an aqueous suspension comprising (a)reversible clusters of drug loaded nano-resin particles, said clustershave a D₅₀ value of at least 2 micrometers and said drug loadednano-resin particles have a particle size distribution characterized inthat the D₉₀ value is 70 nanometers to 900 nanometers, (b) a suspendingagent.

The present invention was found to be increasing the bioavailabilityand/or prolonging ophthalmic action of a drug, when instilling into theeye, the aqueous suspension of reversible clusters of drug loadednano-resin particles, said clusters having a D₅₀ value of at least 2micrometers and said drug loaded nano-resin particles having a particlesize distribution characterized in that the D₉₀ value is 70 nanometersto 900 nanometers, wherein the aqueous suspension contains a suspendingagent.

The present invention also provides a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, saidmethod comprising instilling into the eye an aqueous suspensioncomprising (a) reversible clusters of drug loaded nano-resin particles,said clusters having a D₅₀ value of at least 2 micrometers and said drugloaded nano-resin particles having a particle size distributioncharacterized in that the D₉₀ value is 70 nanometers to 900 nanometers,and (b) a suspending agent.

The present invention also provides a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, themethod comprising providing an aqueous suspension comprising (a)reversible clusters of drug loaded nano-resin particles, said clustershaving a D₅₀ value of at least 2 micrometers and said drug loadednano-resin particles having a particle size distribution characterizedin that the D₉₀ value is 70 nanometers to 900 nanometers, and (b) asuspending agent.

The ‘drugs’ suitable according to the method of the present inventioninclude therapeutically active ingredients that are capable of forming asalt with an acid or an alkali, and includes ionizable therapeuticallyactive ingredients. According to one aspect, drugs include ionizabledrugs that can form salts with acids, known as cationic drugs. Accordingto another aspect, drugs include ionizable drugs that can form a saltwith a base or an alkali, known as anionic drugs. Non limiting examplesof the drugs according to the present invention include drugs that areused ophthalmically such as but not limited to antiglaucoma agents;antibiotics or anti-infective agents; anti-allergic agents;anti-inflammatory/anti-allergic steroids; non-steroidalanti-inflammatory agents; decongestant; local anaesthetic agents;mydriatic agents. The antiglaucoma agents include group of drugs such asbeta-blockers, carbonic anhydrase inhibitors, alpha-adrenergic agonists,prostaglandins, parasympathomimetics and cholinesterase inhibitors.Non-limiting examples of prostaglandins that may be used include,latanoprost, travoprost, bimatoprost, tafluprost, isopropyl unoprostone,8-isoprostaglandin-E2, and the like and pharmaceutically acceptablesalts thereof; Non-limiting examples of beta-blockers that may be usedinclude, timolol, levobunolol, befundol, metipranolol, carteolol,betaxolol, levobetaxolol, befunolol, labetalol, propranolol, metaprolol,bunalol, esmalol, pindolol, hepunolol, metipranolol, celiprolol,azotinolol, diacetolol, acebutolol, atenolol, isoxaprolol orpharmaceutically acceptable salts thereof; Non-limiting examples ofcarbonic anhydrase inhibitors that may be used include, brinzolamide,dorzolamide, acetazolamide, methazolamide, dichlorophenamide and thelike and pharmaceutically acceptable salts thereof; Non-limitingexamples of alpha-adrenergic agonists that may be used include,brimonidine, dipivefrine, clonidine, and clonidinederivatives—p-aminoclonidine, p-acetoamidoclonidine, apraclonidine andthe like and pharmaceutically acceptable salts thereof; Non-limitingexamples of cholinesterase inhibitors that may be used include,physostigmine, ecothiopate and the like and pharmaceutically acceptablesalts thereof; Non-limiting examples of parasympathomimetics that may beused include pilocarpine, demecarium and the like and pharmaceuticallyacceptable salts thereof. Non-limiting examples of antibiotics oranti-infective agents includes moxifloxacin, besifloxacin, gentamicin,neomycin: erythromycin, ciprofloxacin, polymyxin B, beta-lactamantibiotics, tetracycline, chlortetracycline and the like andpharmaceutically acceptable salts thereof; Anti-allergic agents includesolopatadine emedastine, azelastine, epinastine, levocabastine,bepotastine, pheniramine, chlorpheniramine, epinephrine, proepinephrine,norepinephrine, pyrilamine and the like and pharmaceutically acceptablesalts thereof; Anti-inflammatory/anti-allergic steroids includesdextromethorphan, dexamethasone, prednisolone and the like andpharmaceutically acceptable salts thereof. Non-steroidalanti-inflammatory agents includes ketotifen and the like andpharmaceutically acceptable salts thereof; Decongestant includesoxymetazoline, phenylephrine, naphazoline, antazoline and the like andpharmaceutically acceptable salts thereof; Local anaesthetic agentsincludes proparacaine, lidocaine and the like and pharmaceuticallyacceptable salts thereof; Mydriatic agents includes cyclopentolate andthe like and pharmaceutically acceptable salts thereof. A combination oftwo or more drugs may be used. Other drugs which are anionic drugsincludes diclofenac, bromfenac, sulfacetamide, flurbiprofen, ketorolac,lodoxamide, sulfacetamide, cromolyn, pemirolast or theirpharmaceutically acceptable salts or mixtures thereof. According topreferred aspect, the drug is selected from brimonidine, doxycycline,bromfenac, olopatadine, emedastine, dorzolamide, ciprofloxacin,moxifloxacin, besifloxacin, gentamicin, neomycin, polymyxin B,ketotifen, phenylephrine, pyrilamine, dipivefrin, oxymetazoline,levocabastine, azelastine, epinastine, bepotastine, dipivefrin,naphazoline, apraclonidine, levobunolol, betaxolol, levobetaxolol,timolol, carteolol, dextromethorphan, cyclopentolate, proparacaine,pilocarpine, diclofenac, sulfacetamide, flurbiprofen, ketorolac or theirpharmaceutically acceptable salts or mixtures thereof.

According to one preferred embodiment, drug is brimonidine or itspharmaceutically acceptable salt thereof. According to more preferredembodiment, drug is brimonidine tartarate. Brimonidine tartrate ischemically known as 5-bromo-6-(2-imidazolidinylideneamino) quinoxalineL-tartrate. Brimonidine is an alpha adrenergic agonist that reduces theelevated intraocular pressure (IOP) of the eye associated with glaucoma.Brimonidine or its pharmaceutically acceptable salt is present in theaqueous suspension of the present invention in therapeutically activeamounts. In preferred embodiments, brimonidine or its pharmaceuticallyacceptable salt is present at a concentration of 0.05% to 0.5% weight byvolume. In one specific embodiment, brimonidine tartrate is present at aconcentration of 0.35% weight by volume. In another embodiment, theaqueous suspension of the present invention contains brimonidinetartrate at a concentration of 0.15% weight by volume.

According to one aspect of the invention, the resin is an ion exchangeresin. The ion exchange resins are covalently bound in repeatingpositions on the resin chain. These charged groups associate with otherions of opposite charge. Depending on whether the mobile counterion is acation or an anion, it is possible to distinguish between cationic andanionic exchange resins. The matrix in cationic exchangers carries ionicgroups such as sulfonic, carboxylate and phosphate groups. The matrix inanionic exchangers carries primary, secondary, tertiary or quaternaryammonium groups. The resin matrix determines its physical properties,its behavior towards biological substances, and to a certain extent, itscapacity. Since cationic drugs such as brimonidine have a positivecharge, they can bind with cation exchange resins. Sulfonic acidexchangers are the most common cation exchange resins used for drugresinate aqueous suspension of the present invention. In general, theyare cross-linked polystyrenes with sulfonic acid groups which have beenintroduced after polymerization by treatment with sulfuric acid orchlorosulfonic acid. Suitable cation exchange resins that may be used inthe present invention includes, but are not limited to, sodiumpolystyrene divinyl benzene sulphonate, such as marketed by Rohm andHaas, under the trade name Amberlite™ IRP 69; polacrilex resin which isderived from a porous copolymer of methacrylic acid and divinylbenzene,such as marketed by Rohm and Haas, under the trade name Amberlite™ IRP64; polacrilin potassium, which is a potassium salt of a cross linkedpolymer derived from methacrylic acid and divinylbenzene, such asmarketed by Rohm and Haas, under the trade name Amberlite™ IRP 88. Theresins marketed by the company Ion Exchange India Ltd., under thetradenames such as INDION™234; INDION™264; INDION™ 204; INDION™ 214 mayalso be used. In one embodiment, the preferred resin used in the presentinvention is Amberlite IRP69 which is derived from a sulfonatedcopolymer of styrene and divinyl benzene. Amberlite IRP-69 is apharmaceutical grade strong cation exchange resin and structurally apolystyrene sulfonic acid resin cross-linked with divinyl benzene, i.e.polystyrene divinyl benzene sulfonate. Amberlite IRP-69 resin isavailable commercially from Rhom & Haas Company. The mobile orexchangeable cation in the resin is sodium, which can be exchanged for,or replaced by, cationic (basic) species. In embodiments of the ionexchange delivery system of the present invention, positively chargedcationic drug is bound to the negatively charged sulfonic acid groups ofthe Amberlite resin.

Suitably, in some embodiments of the present invention, the ion-exchangeresin is an anion exchange resin and the drug is anionic in nature.Non-limiting example of anionic ionizable drugs include diclofenac,bromfenac, sulfacetamide, flurbiprofen, lodoxamide, sulfacetamide,cromolyn, pemirolast, ketorolac or their pharmaceutically acceptablesalts or mixtures thereof. The matrix in anionic exchange resingenerally carries primary, secondary, tertiary or quaternary ammoniumgroups. Suitable anion exchange resins that may be used in the presentinvention includes, but are not limited to, cholestyramine resin, suchas marketed by Rohm and Haas, under the trade name Duolite™ AP143/1093;INDION™860, which is a macroporous weakly basic anion resin having atertiary amine functionality attached to a polymeric styrene divinylbenzene matrix; INDION™GS400, which is strong base Type II anionexchange resin, based on cross linked polystyrene matrix with benzyldimethyl ethanol amine functional groups.

The amount of ion exchange resin present in the aqueous suspension ofthe present invention, may range from about 0.05% to 5.0% weight byvolume of the suspension. The weight ratio of resin to drug may rangefrom 0.1:1 to 1:0.1, more preferably from 0.3:1 to 1:0.3. In onepreferred embodiment, the weight ratio between the nano-resin particlesand drug is about 1:1. Nanoresin particles used according to the presentinvention have a particle size distribution characterized in that theD₉₀ value is 70 nms to 900 nms, preferably 200 nms to 700 nms.

The aqueous suspension may contain one or more suspending agents. Thesuspending agents are used to increase the viscosity of the suspension,to cause clustering of the nanoresin particles into micron sizeclusters, and to prevent the caking of the suspension. The suitablesuspending agent is selected from an anionic polymer, a non-ionicpolymer or mixtures thereof. The anionic polymers may be selected fromthe group consisting of polymers of acrylic acid like carboxyvinylpolymer or carbomer, also known as Carbopols. Various grades ofcarbomers including Carbopol 934P, 974, 1342 and the like may be used inthe present invention. The polymers of acrylic acid may be present inthe aqueous suspension of the present invention in an amount rangingfrom about 0.01% to 0.5% weight by volume of the suspension. Otheranionic polymers that can be used include, but are not limited to,sodium hyaluronate: sodium carboxymethylcellulose; guargum; chondroitinsulphate; sodium alginate. Particularly, the preferred anionic polymersthat may be used include Carbopol 974P. This anionic polymer is mostpreferably used in an amount of 0.1% w/v of the suspension. Thenon-ionic polymers that can be used according to the present inventionmay be selected from the group consisting of non-ionic polymers such aspolyvinyl pyrrolidone, soluplus-a polyvinyl caprolacatam-polyvinylacetate-PEG graft co-polymer, poloxamers, polyvinyl alcohol,polypropylene glycol, cellulose derivatives like hydroxyethylcellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethyl cellulose and the like. The non-ionic polymers may bepresent in the aqueous suspension of the present invention in an amountranging from about 0.1% to about 5.0% weight by volume of thesuspension. The preferred non-ionic polymers that may be used includehydroxypropyl methylcellulose and polyvinylpyrrolidone. Variouspharmaceutically acceptable grades of hydroxypropyl methylcellulose(also known as hypromellose or HPMC or Methocel) andpolyvinylpyrrolidine (also known as povidone or PVP or plasdone) may beused. The preferred grades of polyvinylpyrrolidine which can be used inthe suspensions of the present invention include PVP K-30, PVP K-25, PVPK-50: PVP K-60 and PVP K-90. It may be present in the aqueous suspensionin an amount ranging from about 0.5% to about 3.0% weight by volume ofthe suspension. The most preferred grade used in the aqueous suspensionof the present invention is PVP K-90, whose 10% w/v aqueous solution hasa dynamic viscosity in the range of about 300.0 cps to about 700.0 cpsat 20° C., and has an approximate molecular weight of about 1,000,000 to1,500,000. In preferred embodiment, polyvinylpyrrolidine PVP K-90 isused in an amount of 1.2% w/v of the suspension. The preferred grades ofhydroxypropylmethylcellulose which may be selected to be used in theaqueous suspensions of the present invention include, but is not limitedto METHOCEL E, (USP grade 2910/HYPROMELLOSE 2910); METHOCEL F, (USPgrade 2906/HYPROMELLOSE 2906); METHOCEL A15 (Premium LV): METHOCEL A4C(Premium); METHOCEL A15C (Premium); METHOCEL A4M (Premium), HPMC USPGrade 1828 and the like. It may be present in the aqueous suspension ofthe present invention in an amount ranging from about 0.5% to about 3.0%weight by volume of the suspension. In most preferred embodiment, theaqueous suspension comprises Hypromellose 2910 in an amount of 0.3% w/v.As auxillary to the suspending agents the flocculation of nanoresinparticles may also be assisted by electrolytes.

The aqueous suspensions according to the present invention may have aviscosity ranging from about 2 cps to 2000 cps, preferably about 5 cpsto 400 cps. The viscosity of the aqueous suspensions may be measured byknown techniques and instruments such as by Brookfield viscometer, understandard conditions. It is to be noted that the aqueous suspensionsaccording to one embodiment of the present invention, maintains itsviscosity upon instillation into the eye, i.e. the viscosity do notchange substantially upon coming in contact with the eye fluid thatcontains various ions such as sodium, potassium, calcium, magnesium,zinc, chloride, and bicarbonate.

The aqueous suspensions according to the present invention mayadditionally comprise other pharmaceutically acceptable excipients suchas chelating agents, preservatives and adjuvants for preservatives. Theaqueous suspension may further include one or more osmoticagents/tonicity adjusting agents, one or more pharmaceuticallyacceptable buffering agents and/or pH-adjusting agents. These excipientsmay be dissolved or dispersed in a pharmaceutically acceptable aqueousvehicle such as water for injection.

In order to achieve, and subsequently maintain, an optimum pH suitablefor ophthalmic preparations, the aqueous suspensions of the presentinvention essentially contains a pH adjusting agent and/or a bufferingagent in suitable amounts. The preferred range of pH for the aqueoussuspension is about 5.0 to about 8.0, preferably 7.0 to 8.0. The mostpreferred pH is about 7.4. The aqueous suspensions of the presentinvention comprise a pharmaceutically acceptable pH adjusting agent thatmay be selected from the group comprising tromethamine, acetic acid orsalts thereof, boric acid or salts thereof, phosphoric acid or saltsthereof, citric acid or salts thereof, tartaric acid or salts thereof,sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, trometamol, and the like and mixtures thereof. Particularly,preferred pH adjusting agents that may be used in the aqueoussuspensions of the present invention include tromethamine, acetic acid,hydrochloric acid, sodium carbonate and sodium hydroxide, mostpreferably tromethamine.

The aqueous suspensions of the present invention are required to beisotonic with respect to the ophthalmic fluids present in the human eyeand are characterized by osmolalities of 250-375 mOsm/kg. Osmolality isadjusted by addition of an osmotic/tonicity adjusting agent. Osmoticagents that may be used in the suspension of the present invention tomake it isotonic with respect to the ophthalmic fluids present in thehuman eye, are selected from the group comprising sodium chloride,potassium chloride, calcium chloride, sodium bromide, mannitol,glycerol, sorbitol, propylene glycol, dextrose, sucrose, and the like,and mixtures thereof. These are used in suitable amounts to maintaindesired osmolarity. In preferred embodiments of the present invention, anon-ionic osmotic agent such as mannitol is used as the osmotic agent.Mannitol may be present in the suspension of the present invention in anamount ranging from about 2.0% to about 6.0% w/v, preferably from about3.0% to about 5.0% w/v and most preferably in an amount of about 4.5%w/v.

Further, the aqueous suspensions of the present invention may comprisepreservatives in antimicrobial effective amounts. The preservative thatmay be used in the aqueous suspensions of the present invention may beselected from, but not limited to: quaternary ammonium compounds such asbenzalkonium chloride (BKC), benzododecinium bromide, cetrimoniumchloride, polixetonium and benzethonium chloride; organic mercurialssuch as phenylmercuric acetate, phenylmercuric nitrate and thimerosal;parabens such as methyl and propyl paraben; ethyl paraoxybenzoate orbutyl paraoxybenzoate; acids and their pharmaceutically acceptable saltssuch as sorbic acid, potassium sorbate, ascorbic acid, boric acid,borax, salicylic acid; substituted alcohols and phenols such aschlorobutanol, benzyl alcohol, phenyl ethanol: amides such as acetamide:other preservatives like Polyquado, polyhexamethylene biguanide, sodiumperborate, aminomethyl propanol, chlorhexidine acetate, self preservedsystem containing ionic preservative like combination of zinc andborate: and the like, and combinations thereof. Preferably theophthalmic aqueous suspensions of the present invention comprise‘quaternary ammonium compound’ as a preservative, particularlybenzalkonium chloride. Benzalkonium chloride is characterized as amixture of alkyldimethylbenzylammonium chlorides. It is employed in theaqueous suspension of the present invention in a concentration of about0.005 to about 0.05% w/v, preferably 0.02% w/v. The aqueous suspensionsmay further comprise an adjuvant to a preservative in suitable amounts,such as N-lauroyl sarcosine sodium. Suitable chelating agent that may beused is edetate disodium.

The aqueous suspension of the present invention remains physically andchemically stable during the shelf life of the product. For instance, incase of aqueous suspensions containing the drug brimonidine, thereoccurs no significant change in assay of brimonidine after long termstorage of the suspension. The assay of the drug remained withinspecified limit and no degradation products or impurities were observedupon storage. Also, there was no significant increase in relatedsubstances.

It was found from the tissue distribution study that the methodaccording to the present invention is capable of delivering a drug tothe posterior segment of the eye. The ability of the present inventionto deliver the drug in the posterior segment of the eye make it suitablefor treating diseases of the posterior segment, which are difficult totreat. For instance in case of brimonidine, delivery to posteriorsegment, helps in preventing degeneration of neurons and impartingneuroprotection action. This was an indeed a surprising finding becausethe method provided not only increased bioavailability of drugs alongwith prolonged ophthalmic action, upon instillation into the eye, butalso could achieve delivery to the posterior segment. Without wishing tobe bound by any theory, it is believed that the reversible cluster ofthe drug loaded nano-resin particles upon instillation into the eye,declusters under shear due to eye blinking. The declustered individualdrug-loaded nano-resin particles spread on the surface of the cornea andsubsequently releases the drug through ion exchange phenomenon under theeffect of ions present in the ocular tissue. There occurs decrease innaso-lacrymal drainage of the drug. The overall phenomenon causesincreased bioavailability of the drug along with prolonged ophthalmicaction.

In preferred embodiments, the present invention provides a method ofincreasing the bioavailability and/or prolonging the ophthalmic actionof a drug, the method comprising instilling into the eye an aqueoussuspension comprising reversible clusters of drug loaded nano-resinparticles, said clusters have a D₅₀ value of at least 2 micrometers andsaid drug loaded nano-resin particles have a particle size distributioncharacterized in that the D₉₀ value is 70 nanometers to 900 nanometers,wherein the nano-resin is selected from a cation exchange or an anionexchange resin, wherein the aqueous suspension further comprises asuspending agent selected from a non-ionic polymer, an anionic polymeror mixtures thereof.

In preferred embodiments, the present invention provides a method ofincreasing the bioavailability and/or prolonging the ophthalmic actionof a drug, the method comprising instilling into the eye an aqueoussuspension comprising reversible clusters of drug loaded nano-resinparticles, said clusters having a D₅₀ value of at least 2 micrometersand said drug loaded nano-resin particles having a particle sizedistribution characterized in that the D₉₀ value is 70 nanometers to 900nanometers, wherein the nano-resin is selected from a cation exchange oran anion exchange resin, wherein the aqueous suspension furthercomprises a suspending agent selected from a non-ionic polymer, ananionic polymer or mixtures thereof, and wherein the drug is brimonidinetartarate. Brimonidine tartrate is present at a concentration rangingfrom about 0.05% w/v to 0.5% w/v of the aqueous suspension. The weightratio between the nano-resin particles and brimonidine may range fromabout 0.1:10 to 10:0.1, preferably from 0.1:1 to 1:0.1, more preferably0.3:1 to 1:0.3. In one preferred embodiment, the weight ratio betweenthe nano-resin particles and brimonidine is about 1:1. In one specificembodiment, the ion exchange resin is polystyrene divinyl benzenesulfonate and it is present at a concentration ranging from about 0.05%w/v to 0.5% w/v of the aqueous suspension.

In a more particularly preferred embodiment, the present inventionprovides an aqueous suspension comprising (a) reversible clusters ofdrug loaded nano-resin particles, said clusters having a D₃₀ value of atleast 2 micrometers and said drug loaded nano-resin particles having aparticle size distribution characterized in that the D₉₀ value is 70nanometers to 900 nanometers, and (b) a suspending agent; wherein thedrug is brimonidine tartrate and it is present in an amount of 0.35%w/v; resin is polystyrene divinyl benzene sulfonate; suspending agent isa mixture of carbopol, polyvinylpyrrolidone and hydroxypropylmethylcellulose. The weight ratio between the nano-resin particles andbrimonidine is about 1:1; and pH is about 7 to 8. Further, surprisingly,the aqueous suspension of the present invention in spite of containinghigher concentration (about 0.35% w/v) of the drug, provided aophthalmic suspension that was very safe with no adverse effects ortoxicity. This result was indeed surprising because inspite ofinstilling the aqueous suspension for consecutive 14-day in New Zealandwhite rabbits, no adverse effects were observed. The results alsoprovided unexpected finding that even at an ocular dosing as high as sixtimes of the desired dose, there was no adverse effects in the eye suchas local toxicity at the site of application or systemic toxicity.Further, there were no signs of irritation, swelling or redness, orallergic reactions.

According to this aspect, the aqueous suspension of the presentinvention was found to increase the bioavailability as well asprolonging ophthalmic action of brimonidine tartrate at a concentrationof 0.35% weight by volume when instilled into the eye once-a-day. Thisprovided an effective use in the treatment of glaucoma by once-a-dayinstillation of the aqueous suspension into the eyes. The method ofincreasing the bioavailability and/or prolonging ophthalmic actionaccording to this embodiment was found to provide equivalent efficacy tothat of Alphaghan P®, but at a reduced frequency of administration thatis, once daily instillation as compared to thrice daily instillation asprescribed for Alphagan P®. (illustrated in Example 8 of thespecification). The method of the invention also advantageously provideoptimum efficacy at a reduced dose. For instance, in this embodiment, areduced (lower) daily dose is administered in case of once dailyadministration of 0.35% w/v suspension of the present invention ascompared to thrice daily administration of 0.15% w/v formulation ofAlphagan P®, but still equivalent efficacy is obtained.

In another preferred embodiment, the present invention provides anaqueous suspension comprising (a) reversible clusters of drug loadednano-resin particles having a particle size distribution characterizedin that the D₉₀ value is 70 nanometers to 900 nanometers, said clustershaving a mean size (D₅₀ value) of at least 2 micrometers, and (b) asuspending agent wherein the drug is brimonidine tartrate and it ispresent in an amount of 0.15% w/v; resin is polystyrene divinyl benzenesulfonate; suspending agent is a mixture of carbopol,polyvinylpyrrolidone and hydroxypropylmethyl cellulose. The weight ratiobetween the nano-resin particles and brimonidine is about 1:1: and pH isabout 7 to 8. The aqueous suspension when instilled twice a day wasfound to provide equivalent efficacy to that of Alphaghan P®, which isinstilled thrice a day. This has been established and provided inExample 9 of the patent specification. According to this aspect, thepresent invention thus provides a method of increasing thebioavailability and/or prolonging ophthalmic action of the drug,comprising providing the aqueous suspension described above. The methodof the invention advantageously provide optimum efficacy at a reduceddaily dose. For instance, in this embodiment, a reduced (lower) dailydose is administered in case of twice daily administration of 0.15% w/vsuspension of the present invention as compared to thrice dailyadministration of 0.15% w/v formulation of Alphagan P®, but stillequivalent efficacy is obtained.

According to one particular embodiment, the aqueous suspension of thepresent invention comprises an ionizable antiglaucoma drug for examplebrimonidine and an additional antiglaucoma drug selected from the groupconsisting of beta-blockers, carbonic anhydrase inhibitors,alpha-adrenergic agonists, prostaglandins, parasympathomimetics andcholinesterase inhibitors. Non-limiting examples of prostaglandins thatmay be used include, latanoprost, travoprost, bimatoprost, tafluprost,isopropyl unoprostone, 8-isoprostaglandin-E2, or salts thereof.Non-limiting examples of beta-blockers that may be used include,timolol, levobunolol, befundol, metipranolol, carteolol or saltsthereof. Non-limiting examples of carbonic anhydrase inhibitors that maybe used include, brinzolamide, dorzolamide, acetazolamide,methazolamide, dichlorophenamide or salts thereof. Non-limiting examplesof alpha-adrenergic agonists that may be used include, brimonidine,apraclonidine, dipivefrine. Non-limiting examples of cholinesteraseinhibitors that may be used include, physostigmine, ecothiopate. Inpractice, the anti-glaucoma drug forms between 0.1% and 1.0% by weightof the composition.

In one preferred embodiment, the aqueous suspension of the presentinvention comprises a combination of ionizable antiglaucoma drugsbrimonidine and timolol or their salts. In this embodiment, the aqueoussuspension comprises brimonidine tartrate and timolol maleate intherapeutically effective amounts suitable for reducing the elevatedintra-ocular pressure and treating glaucoma. In one specific embodiment,the aqueous suspension of the present invention comprise brimonidinetartrate in an amount ranging from 0.2% w/v to 0.35% w/v and timololmaleate in an amount of 0.1 to 0.5% w/v, wherein the suspension issuitable to provide desired therapeutic efficacy by once-a-day topicalinstillation to the eye. In another specific embodiment, the aqueoussuspension of the present invention comprise brimonidine tartrate in anamount of about 0.1% w/v and timolol maleate in an amount of about 0.5%w/v and wherein the suspension is suitable to provide desiredtherapeutic efficacy by twice-a-day topical instillation to the eye.

In another preferred embodiment, the present invention provides a methodof increasing the bioavailability and/or prolonging ophthalmic action ofa drug, the method comprising instilling into the eye an aqueoussuspension comprising reversible clusters of drug loaded nano-resinparticles, said clusters having a mean size of at least 2 micrometers,said drug loaded nano-resin particles having a particle sizedistribution characterized in that the D₉₀ value is 70 nanometer to lessthan 900 nanometers, wherein the drug is bromfenac sodium; and thenano-resin is an anion exchange resin, preferably Indion™860. Accordingto this aspect, the present invention also provides a method ofprolonging ophthalmic action of bromfenac sodium, comprisingadministering to the eyes, the aqueous suspension described above.

In another preferred embodiment, the present invention provides a methodof increasing the bioavailability and/or prolonging ophthalmic action ofa drug, the method comprising instilling into the eye an aqueoussuspension comprising reversible clusters of drug loaded nano-resinparticles, said clusters having a mean size of at least 2 micrometers,said nano-resin particles having a particle size distributioncharacterized in that the D₉₀ value is 70 nanometer to less than 900nanometers, wherein the drug is doxycycline hyclate; and the nano-resinis an cation exchange resin, preferably sodium polystyrene divinylbenzene sulfonate.

While the present invention is disclosed generally above, additionalaspects are further discussed and illustrated with reference to theexamples below. However, the examples are presented merely to illustratethe invention and should not be considered as limitations thereto.

Comparative Example 1

The resin ‘Amberlite® IRP 69’ was washed with absolute alcohol multipletimes. The resin was further washed with Millipore water until a pHclose to neutral (pH 7.0) was attained. The particle size distributionof the resin was measured using Malvern Mastersizer 2000 Ver.5.60,Malvern Instruments Ltd., Malvern, UK. The histogram of the resin isdepicted in FIG. 1. The resin has particle size distribution such thatD₁₀ is 2.341 micron, D₅₀ is 5.175 micron and D₉₀ is 10.41 micron. Theresin was used for preparation of the composition of Table 1.

TABLE 1 Aqueous suspension of cationic drug: Brimonidine Tartrate withAmberlite resin IRP69 of average particle size of 5 microns S. No.Ingredients Amount (% w/v) 1 Brimonidine Tartrate 0.35 2 Amberlite IRP69 0.35 3 Hydroxy propyl methyl cellulose 0.3 4 Polyvinylpyrrolidone 1.25 Carbopol 974P (carbomer) 0.1 6 Benzalkonium Chloride 0.02 7 EdetateDisodium 0.1 8 N-Lauroylsarcosine sodium 0.06 9 Mannitol 4.5 10Tromethamine q.s to adjust pH to 7.4 0.32 11 Water for Injection q.s.

In a stainless steel (SS 316) beaker, 15% water for injection of totalbatch size was taken and heated to 85° C. Hydroxy propyl methylcellulose (hypromellose 2910) was dispersed with high speed stirring toobtain uniform dispersion. The stirring was continued till temperaturereached 25° C. In another stainless steel (SS 316) beaker, a portion ofwater for injection of total batch size was taken at 25° C.Polyvinylpyrrolidone (povidone K-90) was dispersed in water forinjection with stirring to obtain uniform dispersion.

In a stainless steel (SS 316) beaker, about 10% water for injection oftotal batch size was taken and heated at 65° C. Carbopol 974P wasdispersed in heated water for injection with stirring. The stirring wascontinued till the temperature reached 25° C. The Carbopol 974P slurrywas neutralized (pH7.4) with tromethamine. The hypromellose and povidonepolymer dispersions obtained above were added sequentially to thecarbopol 974P phase. The polymer mixture was autoclaved at 121° C. for20 minutes. N-lauryl sarcosine sodium was mixed in a portion of waterfor injection of total batch size and added to the polymer phase afterfiltration through 0.2 micron nylon filter. Mannitol was dissolved in aportion of water for injection at 50-60° C. and benzalkonium chloride,and edetate disodium were added to form a clear solution. This solutionwas added to the above polymer phase. 15% water for injection of totalbatch size was taken in a vessel and Amberlite IRP 69 was dispersed withstirring. In another vessel, 15% water for injection of total batch sizewas taken and brimonidine tartrate was added with stirring to dissolve.This solution was filtered through 0.2 micron and 0.45 micron nylonfilter. Filtered brimonidine tartrate solution was added to aboveautoclaved amberlite IRP 69 dispersion and stirred for 30 minutes. TheAmberlite IRP 69 & Brimonidine tartrate dispersion was added to thepolymer mixture obtained above with stirring and stirring was continuedfor 1 hr. The pH was adjusted with tromethamine solution to about 7.4.The volume of suspension was finally made up to 100% batch size. Thesuspension was stirred for 60 minutes, followed by homogenization at15000 rpm for 10 mins.

The suspension was subjected to sonication at a frequency of 33±3 KHz,for 5 seconds and a sample was withdrawn to measure the particle sizedistribution by Malvern Mastersizer 2000. Ver.5.60, Malvern InstrumentsLtd., Malvern, UK. The histograms of the particle size distribution uponapplication of first pulse of shear/sonication for 5 seconds is depictedby FIG. 2. Following intervals of one minute each, the sonicationprocess and subsequent measurement of the particle size was repeatedtill 5 minutes. FIGS. 3 to 7, are the histograms of the particle sizedistribution at the end of each minute, till 5 minutes, respectively.See table number 2 for the particle size distribution.

TABLE 2 Effect of shear on the particle size distribution of the resinparticles in the suspension Volume mean diameter in microns recorded byMalvern lazer diffraction method Example No. PSD* Initial 1 min 2 min 3min 4 min 5 min Figure. No. FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7Comparative D₁₀ 1.587 1.380 1.285 1.351 1.332 1.348 example 1 D₅₀ 5.6215.200 5.088 5.160 5.112 5.176 D₉₀ 13.523 10.943 10.861 10.836 10.76010.883 PSD*—Particle Size Distribution in Volume mean diameter inmicrons

Observation: It was observed that particle size distribution of the drugloaded resin particles remains in the microns size, in that the Ds was 5microns at the end of 5 minutes, after application of shear bysonication at a frequency of 33±3 KHz, for 5 seconds. Further, the D₉₀was also not affected by the shear force, and remained in the range of10 microns.

Example 1

Preparation of purified nanoresin: The resin ‘Amberlite® IRP69’ waswashed multiple times with a suitable alcohol such as methanol orabsolute alcohol. The resin was further washed with heated Milliporewater until a pH close to neutral (pH 7.0) was attained. The washedresin was subjected to wet milling to reduce particle size to nanometerrange, having D₉₀ less than 900 nms. The washed resin and stabilizedZirconia beads were added to water for injection in a vessel containinga teflon coated magnetic bead. The vessel was kept for wet grinding on amagnetic stirrer for about 24-48 h to obtain nano size milled resinparticles. The slurry so formed was passed through a 25 micron sieve toremove beads and further passed through 40 micron PP filter. The milledresin suspension obtained above was subjected to diafiltration using a500 kD hollow fiber cartridge wherein the water extractable impuritieswere reduced to less than 1.0% by weight of resin. The milled resinsuspension was further washed with water for injection. This slurry waslyophilized to get the dried powder form of the milled purified resin.

The particle size distribution of the milled resin was measured usingMalvern Mastersizer 2000 Ver.5.60, Malvern Instruments Ltd., Malvern,UK. The histogram of the resin is depicted in FIG. 8. The particle sizedistribution was such that D₁₀=0.148 microns, D₅₀=0.24 microns andD₉₀=0.606 microns. The nanoresin was used for the preparation of aqueoussuspension in Example 2 to Example 5.

Example 2(A) and 2(B)

TABLE 3 Details of the aqueous suspension of the present inventionAmounts- % w/v Ingredients Example Example function Ingredients 2(A)2(B) Active ingredient Brimonidine Tartrate 0.35 0.15 Resin AmberliteIRP 69 0.35 0.15 Polymeric vehicle Hydroxy propyl methyl 0.3 0.3cellulose Polymeric vehicle Polyvinylpyrrolidone 1.2 1.2 Polymericvehicle Carbopol 974P (carbomer) 0.1 0.1 Preservative BenzalkoniumChloride 0.02 0.02 Chelating agent Edetate Disodium 0.1 0.1 PreservativeN-Lauroylsarcosine sodium 0.06 0.06 Osmotic agent Mannitol 4.5 4.5 pHadjusting agent Tromethamine q.s to adjust 0.32 0.32 pH to 7.4 VehicleWater for Injection q.s. q.s.

The aqueous suspension of Example 2 (A) and (B) were prepared as below:In a stainless steel (SS 316) beaker, about 15% water for injection oftotal batch size was taken and heated to 85° C. The specified polymericvehicle, such as hydroxy propyl methyl cellulose (hypromellose 2910) wasdispersed with high speed stirring to obtain uniform dispersion. Thestirring was continued till temperature reached 25° C. In anotherstainless steel (SS 316) beaker, about 12% water for injection of totalbatch size was taken at 25° C. Polyvinylpyrrolidone (povidone K-90) wasdispersed in water for injection with stirring to obtain uniformdispersion. In case of Example 2, in a stainless steel (SS 316) beaker,about 10% water for injection of total batch size was taken and heatedat 65° C. Carbopol 974P was dispersed in heated water for injection withstirring. The stirring was continued till the temperature reached 25° C.The Carbopol 974P slurry was neutralized (pH7.4) with tromethamine. Thehypromellose and povidone polymer dispersions obtained above were addedsequentially to the carbopol 974P phase. The polymer mixture wasautoclaved at 121° C. for 20 minutes. N-lauryl sarcosine sodium wasmixed in a portion of water for injection and added to the polymer phaseafter filtration through 0.2 micron nylon filter. Mannitol was dissolvedin a portion of water for injection at 50-60° C. and benzalkoniumchloride, and edetate disodium were added to form a clear solution. Thissolution was added to the above polymer phase. A portion of water forinjection of total batch size was taken in a vessel and Amberlite IRP 69obtained as per Example 1, was dispersed with stirring. This dispersionwas autoclaved at 121° C. for 20 minutes. In another vessel, a portionof water for injection was taken and brimonidine tartrate was added withstirring to dissolve. This solution was filtered through 0.2 micron and0.45 micron nylon filter. Filtered brimonidine tartrate solution wasadded to above autoclaved Amberlite IRP 69 dispersion and stirred for 30minutes.

The Amberlite IRP 69 & Brimonidine tartrate dispersion was added to thepolymer mixture obtained above with stirring and stirring was continuedfor about 30 minutes to 1 hour. The volume of suspension was finallymade up to 100% batch size. The suspension was stirred for about 60minutes, followed by homogenization at 15000 rpm for 10 mins. The pH wasadjusted with tromethamine solution to about 7.4. The viscosity ofaqueous suspension of Example 2(A) was measured using Brookfieldviscometer and it was found to be 19.7 cps.

Example 3-5

The aqueous suspensions of Example 3 to 5 were prepared in a similarmanner as above but excluding steps of addition of HPMC and PVP.

TABLE 4 Details of the aqueous suspension of the present invention % w/vIngredients Exam- Exam- Exam- function Ingredients ple 3 ple 4 ple 5Active ingredient Brimonidine Tartrate 0.35 0.35 0.35 Resin AmberliteIRP 69 0.35 0.35 0.35 Polymeric vehicle Carbopol 974P 0.1 0.2 0.3(carbomer) Preservative Benzalkonium 0.02 0.02 0.02 Chloride Chelatingagent Edetate Disodium 0.1 0.1 0.1 Preservative N-Lauroylsarcosine 0.060.06 0.06 sodium Osmotic agent Mannitol 4.5 4.5 4.5 pH adjusting agentTromethamine q.s to 0.3 0.3 0.3 adjust pH to 7.4 Vehicle Water forInjection q.s. q.s. q.s.

The viscosity of aqueous suspension of Example 3 to 5 was measured usingBrookfield viscometer. The viscosity was found to be 8.2 cps, 12.0 cpsand 97.9 cps respectively.

Example 6

Evaluation of chemical stability was made, for which the aqueoussuspension of Example 2(A) was filled in 5 ml white opaque LDPEcontainers. The bottles filled with suspension of Example 2(A) weresubjected to accelerated stability conditions to determine storagestability during the shelf life of the product. The bottles were kept atdifferent conditions. The bottles were kept in upright as well as ‘onthe side’ position. The observation for assay of brimonidine is givenbelow:

TABLE 5 Results of the stability data of the suspension stored inbottles in upright position 25° C./ 30° C./ 40° C./ 40% RH 35% RH 25% RHAssay Initial 6 M 6 M 6 M % Brimonidine tartrate 101.1 99.58 96.11 96.91

TABLE 6 Results of the stability data of the suspension stored inbottles & kept ‘on the side’ position 25° C./ 30° C./ 40° C./ 40% RH 35%RH 25% RH Assay Initial 6 M 6 M 6 M % brimonidine tartrate 101.1 99.2798.01 95.74

The results in Table 5 and Table 6 indicate that there was nosignificant change in assay of brimonidine after long term storage. Theassay of the drug remained within specified limit and no degradationproducts or impurities were observed upon storage. Also there was nosignificant increase in related substances. The suspension according tothe present invention remained chemically stable during the shelf lifeof the product. The suspension is room temperature stable.

Example 7

The example describes the effect of shear on the reversible clusters ofdrug loaded nano-resin particles suspended in Example 2(A), whichdecluster into individual drug-loaded nano-resin particles whensubjected to shear, such as a shear resulting from blinking in the eye.This effect was measured in terms of particle size distribution,initially and upon application of shear.

Procedure: The test samples were subjected to shear by placing the vialscontaining the suspension on bath sonicator (Model type: MC-109 and SIno—1909: Mfg. by Oscar Ultrasonic Pvt. Ltd.) and shear was applied inthe form of sonication frequency of 33±3 kHz for 5 seconds and thesample was withdrawn to measure the particle size distribution.Following intervals of 1 minute each the process is repeated 5 times.

The particle size measurement was done using Malvern Mastersizer 2000,Ver.5.60, Malvern Instruments Ltd., Malvern, UK, but the analyser'ssonication means were not used. The sample was only subjected to mildstirring by a mechanical stirrer. The observations are summarized belowin Table 7.

TABLE 7 Effect of shear on the particle size distribution of the resinparticles: Volume mean diameter in microns recorded by Malvern lazerdiffraction method Example No. PSD* Initial 1 min 2 min 3 min 4 min 5min Figure. No. FIG. 9 FIG. 10 FIG. 11 FIG. 12 FIG. 13 FIG. 14Example2(A) D₁₀ 0.852 0.475 0.338 0.151 0.145 0.140 D₅₀ 19.549 13.8820.996 0.254 0.229 0.213 D₉₀ 58.970 49.111 30.405 1.398 0.512 0.449PSD*—Particle Size Distribution in Volume mean diameter in microns

The histograms of the particle size distribution for suspension ofExample 2(A) before sonication is depicted by FIG. 9. The suspension wasthen subjected to sonication at a frequency of 33±3 KHz, for 5 secondsand a sample was withdrawn to measure the particle size distribution.The histogram of the particle size distribution upon application offirst pulse of shear/sonication for 5 seconds is depicted by FIG. 10.Following intervals of one minute each, the sonication process andsubsequent measurement of the particle size by Malvern, was repeatedtill 5 minutes.

FIGS. 11 to 14, are the histograms of the particle size distribution atthe end of each minute, till 5 minutes, respectively. See above tablenumber 7 for the particle size distribution.

Observations: It was found that clusters of drug loaded nano-resinparticles of Example 2(A), disintegrated completely as shear was appliedto the suspension. This was evident by the decrease in the particle sizeobserved upon application of shear/sonication as shown in Table 7 andFIGS. 9 to 14. The D₅₀ of drug-resin nanoparticles was initially about19.5 microns, which upon application of shear at regular interval for 5minutes disintegrated and converted into individual drug-resinnanoparticles having D₅₀ of 0.213 micron (213 nm).

FIGS. 9-14 demonstrates that particles less than 900 nms always remains,which are particles of individual drug-resin particles, while at thesame time there are clusters of drug loaded nano-resin particles havingD₅₀>2 microns, which deagglomerate into individual nano-resin particlesupon application of shear. FIG. 13-14 which corresponds to applicationof shear at 4 and 5 minutes represents largely individual nano-resinparticles and for all the purposes of the specification, are taken torepresent the particle size distribution of the individual drug loadednano-resin particles.

On the contrary, in case of Comparative Example 1 there was nodisintegration and the particle size did not change. This is apparentfrom Table 2 and FIGS. 2 to 7. The D₅₀ of the suspended drug-resinparticles was initially about 5.2 microns and even after application ofshear at regular interval for 5 minutes, the size of the particles didnot changed and remained almost the same, viz. 5.176 microns.

Example 8

The efficacy (intraocular pressure reduction effect) of the suspensionof Example 2(A), administered once a day, was tested in New Zealandwhite (NZW) rabbits. It was compared with Alphagan®P, 0.15% w/v, whichwas administered three times a day and placebo which was administeredonce a day. Ocular hypertension was induced unilaterally (i.e. in lefteye, right eye as a control) in male NZW rabbits by intravitrealinjection of 0.2 ml dexamethasone (Solodex® eye drop 0.1% w/v), twoinjections given 10 days apart. Significant increase in intraocularpressure (>6 mmHg) compared to contralateral eye was established fromday 20 of first injection of dexamethasone sustaining upto day 60; theeffect of treatment with brimonidine suspension on intraocular pressurewas evaluated during this period. Animals were randomized on the basisof elevated intraocular pressure to 3 different treatmentgroups—suspension of Example 2(A), (test) (0.35% w/v); Alphagan® P,0.15% w/v and placebo; each group having 5 animals. The duration of doseadministration was 21 days.

35 μl of aqueous suspension (0.35% w/v) of Example 2(A) and 35 μl ofplacebo were instilled once-a-day (8 a.m.) and 35 μl of referenceformulation Alphagan®P (brimonidine tartrate aqueous solution, 0.15%w/v) was instilled three-times-a-day (8 a.m., 2 p.m., and 8 p.m.) for 21consecutive days. Intra ocular pressure in each eye of each animal wasmeasured three times a day at 7 a.m. {i.e. 23 hours post dose of Example2(A), and 11 hour post dose of Alphagan®P, represented in the figure by‘0 hr’}; 10 a.m., (2 hr post dosing) and 1 p.m. (5 hr post dosing) onday 1, 3, 4, 7, 10, 13, 16, 19 and 21 during the 21-day treatmentperiod. After discontinuation of the treatments the intra ocularpressure was measured at 7 a.m. on day 22 and at 7 a.m. and 10 a.m., onday 23. Observations for change in intra ocular pressure at differenttime points are presented in FIG. 18.

Observations and inference: Topical delivery of aqueous suspension ofExample 2(A) to the eyes, when administered once-a-day, causedstatistically significant reduction in intra ocular pressure whenmeasured at approximately 48 hours (day 3) of its first dose. Asignificant reduction in intra ocular pressure was observed till day 22i.e. 24 hours after the last dose.

Topical instillation of Alphagan® P (0.15%, three times a day) causedstatistically significant reduction in IOP when measured at day 7 of itsfirst dose (i.e. 12 hrs after the last dose of day 6). A significantreduction in IOP was observed till day 21 (i.e. 5 hrs after the firstdose of day 21). Reduction in IOP was not statistically significant inAlphagan®P treated group on day 13, 16, 19, and 21 at approx. 12 hrsafter the last dose of previous day. It may be concluded that aqueoussuspension of the present invention provided prolonged intra ocularpressure lowering effect in once-a-day dosing schedule. This effect wascomparable with three times-a-day dosing of Alphagan® P (brimonidinetartrate aqueous solution, 0.15%).

Example 9

The example illustrates the method of prolonging ophthalmic action ofdrug ‘brimonidine tartrate’ to the eye, said method comprisingadministering the aqueous suspensions of the present invention. Theefficacy (intraocular pressure reduction effect) of the suspension ofExample 2(A) (having 0.35% w/v of brimonidine tartrate), administeredonce a day, as well as another suspension of present invention Example2(B) (having 0.15% w/v of brimonidine tartrate), administeredtwice-a-day was tested in NZW rabbits. It was compared with Alphagan® P,0.15% w/v, which is administered three times a day.

Male NZW Rabbits [5-8 months (at the time of receipt); 1.4-3.2 kg] wereused for the study. On day 1, animals were divided into 4 groups asbelow consisting of five animals in each group.

-   -   Placebo    -   Brimonidine Tartrate Aqueous suspension, 0.35% w/v of Example 2        (A)—Test item 1    -   Brimonidine Tartrate Aqueous suspension, 0.15% w/v of Example        2(B)—Test item 2    -   Alphagan® P, 0.15% w/v—Reference formulation

Left eye of each animal was assigned to receive respective drug solution(volume—35 μl) for the 10 days treatment periods (day 3 to day 12).Pretreatment measurements of intra ocular pressure were obtained forboth the eyes of each animal at 8 AM and 6 PM for two days precedingtreatment (day 1 to day 2) and 8 AM on day 3, using PneumatonometerModel 30 Classic™ (Reichert, USA) and averaged intra ocular pressurevalue up to 48 hours were considered as initial (baseline) intra ocularpressure reading. During intra ocular pressure measurements, each animalwas restrained in restrainer without sedation. The pneumatonometer probewas placed lightly on the cornea and allowed to rest for 10-15 seconds.The probe was placed entirely on the cornea in horizontal position andfive consecutive readings were recorded, each with standard deviationvalue <1 which was displayed on the screen. The pneumatonometer probefilter was cleaned after each use by gently touching to cotton swab(immersed in saline) and just wiped with tissue paper. On day 3, 5, 7, 9& 11, intra ocular pressure was measured at 8 AM and immediately afterintra ocular pressure measurement, 35 μl each of placebo; test item 1:test item 2; reference item was instilled intraocularly in left eye ofeach respective assigned animal. Intra ocular pressure readings weremeasured as described above at 10 AM and 2 PM i.e. at 2 hr and 6 hrrespectively post placebo/test/reference item instillation. 35 μlreference item was again instilled at 2 PM to it's group of animals. 35μl of placebo or test item 2 or reference item was also instilled at 8PM to its group's of animals.

On day 4, 6, 8 & 10 at 8 AM, 35 μl of placebo or test item 1 or testitem 2 or reference item was instilled intraocularly in left eye of eachrespective assigned animal. 35 μl reference item was again instilled at2 PM to its group's of animals. 35 μl of placebo or test item 2 orreference item was also instilled at 8 PM to its group's of animals. Onday 13 & 14, intra ocular pressure was measured at 8 AM. The % reductionin intra ocular pressure of test suspensions was calculated with respectto initial (baseline) intra ocular pressure readings of respectivegroup. The observations for change in intra ocular pressure at differenttime points are presented in FIG. 19.

Observation and inference: Once a day intraocular instillation ofaqueous suspension of Example 2(A) (0.35% w/v): and two times a dayinstillation of aqueous suspension of Example 2(B) (0.15% w/v); or threetimes a day instillation of Alphagan® P, showed significant reduction inbaseline intra ocular pressure at 2 hr (10 AM) after first instillationon every treatment day as compared with baseline mean intra ocularpressure value. At 6 hour (2 PM) & 12 hr. (8 AM) post first instillationalso showed intra ocular pressure reduction (but not significant) ascompared with baseline mean intra ocular pressure. There was nostatically significant difference in intra ocular pressure reductionbetween test item 1 or test item 2 or reference item.

Intra ocular pressure reduction potential of aqueous suspension of thepresent invention Example 2(A) (given once-a-day) was comparativelyhigher at both peak (2 hr after instillation) and trough (24 hr afterinstillation) than Alphagan® P (0.15%, TID). Also, the 0.15% brimonidineaqueous suspension, Example 2(B) showed comparative efficacy as comparedto Alphagan® P (0.15%. TID) but at twice daily administration instead ofthree times a day administration as that of Alphagan P® (0.15%).

Example 10

The safety/toxicity profile of aqueous suspension comprising brimonidinetartrate (0.35% w/v) was assessed in New Zealand white rabbits followingdaily ocular administration for 14 days. The aim of the study was toestablish NOAEL i.e. no observed adverse effect levels, exposure levelsand safety criteria for ocular use in humans.

Study Design—

Twenty New-Zealand White rabbits; (10 males and 10 females) wererandomized, based on body weights, into following five study groups.Each group comprised of two animals of both gender.

The desired dose was administered by ocular instillation.

G1 (saline {control}, 360 μL/animal/day), 30 μL per eye/time×6 times adayG2 (Placebo, 360 μL/animal/day), 30 μL per eye/time×6 times a dayG3 (Low dose {test}, 60 μL/animal/day), 30 μL per eye/time×1 times a dayG4 (Mid dose {test}, 180 μL/animal/day): 30 μL per eye/time×3 times adayG5 (High dose {test}, 360 μL/animal/day) 30 μL per eye/time×6 times adayG3, G4 & G5 test=0.35% w/v Brimonidine Tartrate Aqueous Suspension ofthe present invention (Example 2(A))

The test parameters which were evaluated included—Daily Clinical Signsand Mortality; Detailed Clinical Sign Observation; Body Weights;Ophthalmoscopy and Necroscopy. The details of these test parametersalong with the results are described below. Besides this, otherparameters which were also evaluated (but data not given here) include:clinical pathology, histology, biochemistry, prothrombin time and urineanalysis.

Daily Clinical Signs and Mortality—Cage side observations were done,twice daily, for all animals to note clinical signs, adverse effects;including those for eyes, morbidity and mortality during the dosingperiod. These observations were performed once before dosing and postlast dosing between 2-4 hours. Animal check to observe mortality wasperformed twice daily throughout study period and findings wererecorded.

No mortality was observed in control, placebo as well as in test itemdosed groups. During dosing period of 14 days, yellowish exudates(probably clearing of excess test item) staining the areas around botheyes was observed in G4 and G5. No other adverse clinical signs wereobserved.

Detailed Clinical Sign Observation—Detailed observations were performedbefore initiation of dosing and on Days 1, 7 and 14 post dosing. Theanimals were examined closely for clinical signs, general behavior orany other signs. Eyes were examined with hand-held slit lampophthalmoscope and findings were recorded according to Draize scoringsystem described in table 10 below:

TABLE 10 Clinical Sign Observation Corneal opacity: degree of density(the area of corneal opacity should be noted) (maximum possible 4) Noulceration or opacity 0 Scattered or diffuse areas of opacity, detailsof iris clearly visible 1 Easily discernible translucent area, detailsof iris slightly obscured 2 Nacrous area, no details of iris visible,size of pupil barely discernible 3 Opaque cornea, iris not discerniblethrough the opacity 4 Iris (maximum possible 2) Normal 0 Markedlydeepened rugae, congestion, swelling, moderate circum- 1 cornealhyperaemia, or injection, any of these or combination of any thereof itis still reacting to light (sluggish reaction positive) No reaction tolight, hemorrhage, gross destruction (any or all of 2 these)Conjunctivae (maximum possible 3) Redness (refers to palpebral andbulbar conjunctivae, excluding cornea and iris) Normal 0 Some bloodvessels definitely hyperaemic (injected) 1 Diffuse, crimson color,individual vessels not easily discernible 2 Diffuse beefy red 3Chemosis: Swelling (refer to lids and/or nictating membranes) maximumpossible 4 Normal 0 Some swelling above normal 1 Obvious swelling withpartial eversion of lids 2 Swelling with lids about half closed 3Swelling with lids more than half closed 4

During detailed clinical sign observation, no test item related adverseclinical signs were observed in any group throughout the study period.Detailed examination of eyes (including Draize scoring) did not show anyadverse finding/sign. The scoring for all the animals in all groups waszero.

Ophthalmoscopy: Ophthalmoscopy was performed in all animals atinitiation of dosing; thereafter it was performed on Days 7 and 14. Ateach observation, both eyes of animal were examined with hand heldophthalmoscope (Ophthalmoscope Heine). Observations for following werenoted: Eye ball, Lacrimation, Conjunctivae, Eyelids, Sclera, Pupilreaction to light, Cornea, Iris, Anterior chamber, Lens, Vitreous bodyand Fundus with use of mydriatic agent. The Fluorescein-staining ofcornea was done at the end of dosing on Day 14. Examination of corneawas performed with the help of ophthalmoscope.

During ophthalmoscopy, no abnormality was detected in the eye of anyanimal during pre-dose and on Day 7 and 14 at post-dosing. No signs ofcorneal damage or any other abnormality was noticed for cornea withfluorescein strip staining.

Necroscopy—On completion of dosing, all animals from G1 to G5 werenecropsied on day 15. Gross pathology was noted. The cranial, thoracicand visceral cavities were opened and examined macroscopically. Eyeballs, optic nerve and adnexal tissues (eyelids, accessory glands,nictitating membrane, conjunctivae and orbital muscles) were examinedgrossly for any macroscopic change. Microscopic evaluation of tissueswere performed in G1, G2 and G5 and it was not extended to any lowergroup since no test item related histopathological adverse effect wasnoted in G5. Brain, liver, lung with main stem bronchi were peerreviewed in all animals from G1, G2 and G5.

At terminal necropsy, statistically significant increase in absoluteheart weights of G2 males, relative spleen weight in G4 males andrelative adrenal weight in G4 females was noted: however, these changeswere not dose depended, hence not considered as test item relatedadverse effect. Microscopic evaluation of organs/tissues in G2 or G5male and female animals did not show any finding that could be relatedto dosing of placebo or test item. The microscopic findings observed inG2 and G5 were those of incidental/spontaneous nature and comparable tothat in G1. Microscopic examination of eye and its adnexaltissues/organs did not show any test item or placebo related findings.In summary—No mortality was observed for males and females of any dosegroup. During dosing period, yellowish exudates staining around the eyeswas observed both in G4 and G5 which probably was due to clearing out ofexcess test item. No test item related clinical signs were observedduring daily or detailed clinical sign observations. No test itemrelated adverse changes noticed in body weights, percent body weightchanges, ophthalmoscopy, hematology, biochemistry, urine, absolute organweights and relative organ weights of males and females. In males andfemales, no test item related macroscopic or microscopic lesions wereobserved in any organ including eyes in any dose group.

It is concluded that the aqueous suspension of the method of the presentinvention, which contains brimonidine tartarate at a concentration of0.35% weight by volume of the aqueous suspension, when administered as30 μL per eye in both eyes, up to maximum 6 times per day forconsecutive 14 days, did not produce any adverse effects in the eye withno local toxicity at the site of application as well as no systemictoxicity. Thus, the method of the present invention not only provides animproved efficacy in terms of reduction of intraocular pressure but wasalso found to be safe without any adverse effects when administered forprolonged period of time, such as 14 consecutive days or more.

Example 11

The example studies the ocular tissue distribution of ionizable drug,brimonidine tartrate upon instilling into the eye, an aqueous suspensionof the present invention comprising 0.15% w/v of brimonidine tartrate(suspension of Example 2(B); called herein as test formulation), and itwas compared with currently marketed product Alphagan® P having 0.15%w/v of Brimonidine Tartrate (called herein as reference formulation).The study was conducted in New Zealand white rabbits. Values of C_(max)and AUC_(0-t) four hours post instillation (t=4 hours) of the test andreference formulations were determined in ocular tissues of the anteriorand posterior segments. The values of C_(max) (ng·ml⁻¹) and AUC_(0-t)(ng·ml⁻¹·hr⁻¹) comparing the test formulation (present invention) andreference formulation (Alphagan®P) in different tissues (aqueous humour,cornea, sclera, eye-lid, conjunctiva, lens, retina, vitreous humour) arerepresented in FIG. 15 to 17. It can be clearly seen that the maximumconcentration (C_(max)) as well as the bioavailability (AUC_(0-t)) ishigher in all the tissues in case of test formulation, i.e. aqueoussuspension of the present invention versus the reference formulation,i.e. Alphagan®P. The difference in values is significant. For instancein FIG. 15, the C_(max) in cornea for test formulation is 14793 ng·ml⁻¹while it is only 6491 ng·m⁻¹ in case of reference formulation. TheC_(max) in retina for test formulation is 1267 ng·ml⁻¹ while it is only603 ng·ml⁻¹ in case of reference formulation (FIG. 16). Also, a higheramount of drug is reaching tissues in case of test formulation; forinstance the bioavailability. i.e. AUC_(0-t) in the posterior segmenttissue such as retina was 1741 ng·ml⁻¹·hr⁻¹ in case of test formulationversus AUC_(0-t) of 537 ng·ml⁻¹·hr⁻¹ obtained for reference formulation.Thus, from the results of this study which compared equivalent strengths(0.15% w/v) of the test formulation versus the reference formulation, itis evident that the aqueous suspension of the present invention providesincreased bioavailability and higher C_(max) in all tissues, compared toconventional marketed formulation (Alphagan® P).

The present invention thus provides a method of increasing thebioavailability and/or prolonging ophthalmic action of a drug, themethod comprising instilling into the eye an aqueous suspensioncomprising reversible clusters of drug loaded nano-resin particleshaving a particle size distribution characterized in that the D₉₀ valueis 70 nanometer to 900 nanometers said clusters having a mean size of atleast 2 micrometers.

Example 12

TABLE 11 Aqueous suspension according to specific embodiments of thepresent invention comprising a combination of two ionizable drugs,brimonidine tartrate and timolol maleate Ingredients functionIngredients % w/v Active ingredient Brimonidine Tartrate 0.1-0.35 ActiveIngredient Timolol maleate equivalent to timolol 0.5 Resin Sodiumpolystyrene divinyl benzene 0.1-0.35 sulphonate Polymeric vehicleHydroxy propyl methyl cellulose 0.3 Polymeric vehiclePolyvinylpyrrolidone 1.2 Polymeric vehicle Carbopol 974P (carbomer) 0.1Preservative Benzalkonium Chloride  0.02 Chelating agent EdetateDisodium 0.1 Preservative N-Lauroylsarcosine sodium  0.06 Osmotic agentMannitol 4.5 pH adjusting agent Tromethamine q.s to adjust pH to 7.44.2-5.6  Vehicle Water for Injection q.s.

The aqueous suspension was prepared as below:

In a stainless steel (SS 316) beaker, about 15% water for injection oftotal batch size was taken and heated to 85° C. The specified polymericvehicle, such as Hydroxy propyl methyl cellulose (hypromellose 2910) wasdispersed with high speed stirring to obtain uniform dispersion. Thestirring was continued till temperature reached 25° C. In anotherstainless steel (SS 316) beaker, about 12% water for injection of totalbatch size was taken at 25° C. Polyvinylpyrrolidone (povidone K-90) wasdispersed in water for injection with stirring to obtain uniformdispersion. In a stainless steel (SS 316) beaker, about 10% water forinjection of total batch size was taken and heated at 65° C. Carbopol974P was dispersed in heated water for injection with stirring. Thestirring was continued till the temperature reached 25° C. The Carbopol974P slurry was neutralized (pH7.4) with tromethamine. The hypromelloseand povidone polymer dispersions obtained above were added sequentiallyto the carbopol 974P phase. The polymer mixture was autoclaved at 121°C. for 20 minutes. N-lauryl sarcosine sodium was mixed in a portion ofwater for injection and added to the polymer phase after filtrationthrough 0.2 micron nylon filter. Mannitol was dissolved in a portion ofwater for injection at 50-60° C. and benzalkonium chloride, and edetatedisodium were added to form a clear solution. This solution was added tothe above polymer phase. Amberlite IRP 69 was obtained as per Example 1.The particle size distribution of the milled resin was such thatD₁₀=0.074 microns, D₅₀=0.153 microns and D₉₀=0.436 microns. TheAmberlite IRP 69 nanoresin so obtained was dispersed in a portion ofwater for injection with stirring. This dispersion was autoclaved at121° C. for 20 minutes. In another vessel, about 10% of water forinjection of total batch size was taken and brimonidine tartrate wasadded with stirring to dissolve. This solution was filtered through 0.2micron and 0.45 micron nylon filter. Filtered brimonidine tartratesolution was added to above autoclaved Amberlite IRP 69 dispersion andstirred for 30 minutes.

The Amberlite IRP 69 & Brimonidine tartrate dispersion was added to thepolymer mixture obtained above with stirring and stirring was continuedfor 30 minutes. About 10% water for injection of total batch size wastaken and Timolol maleate was added with stirring to dissolve. Thissolution was filtered through 0.2 micron and 0.45 micron nylon filter.Filtered Timolol maleate solution was added to above phase and stir for30 minutes. The pH was adjusted with tromethamine solution to about 7.4.The volume of suspension was finally made up to 100% batch size. Thesuspension was stirred for 60 minutes, followed by homogenization at15000 rpm for 10 minutes.

Example 13

The example provides aqueous suspension formulation of ionizable drugsdoxycycline, according to one embodiment of the present invention

TABLE 12 Doxyclycline suspension, 0.05% w/v Ingredients functionIngredients Quantity % Active Ingredient Doxycycline Hyclate 0.057 (eqto Doxyclycline 0.05%) Resin Sodium polystyrene divinyl benzene 0.019sulphonate (Amberlite IRP 69) Polymeric vehicle Hydroxy propyl methylcellulose 0.3 Polymeric vehicle Polyvinylpyrrolidone 1.2 Polymericvehicle Carbopol 974P (carbomer) 0.1 Preservative Benzalkonium Chloride0.02 Chelating agent Edetate Disodium 0.1 PreservativeN-Lauroylsarcosine sodium 0.06 Osmotic agent Mannitol 4.5 pH adjustingagent Tromethamine q.s to adjust pH to 5.0 0.0248 Vehicle Water forInjection q.s. to 100

The specified polymeric vehicle, such as Hydroxy propyl methyl cellulose(hypromellose 2910) was dispersed with high speed stirring to obtainuniform dispersion. The stirring was continued till temperature reached25° C. In another stainless steel (SS 316) beaker, a portion of waterfor injection was taken at 25° C. Polyvinylpyrrolidone (povidone K-90)was dispersed in water for injection with stirring to obtain uniformdispersion. In a stainless steel (SS 316) beaker, a portion of water forinjection was taken and heated at 65° C. Carbopol 974P was dispersed inheated water for injection with stirring. The stirring was continuedtill the temperature reached 25° C. The Carbopol 974P slurry wasneutralized with tromethamine. The hypromellose and povidone polymerdispersions obtained above were added sequentially to the carbopol 974Pphase. The polymer mixture was autoclaved at 121° C. for 20 minutes.N-lauryl sarcosine sodium was mixed in a portion of water for injectionand added to the polymer phase after filtration through 0.2 micron nylonfilter. Mannitol was dissolved in a portion of water for injection at50-60° C. and benzalkonium chloride, and edetate disodium were added toform a clear solution. This solution was added to the above polymerphase. Amberlite IRP 69 was obtained as per Example 1. The particle sizedistribution of the milled resin was such that D₁₀=0.074 microns,D₅₀=0.153 microns and D₉₀=0.436 microns. The Amberlite IRP 69 nanoresinso obtained was dispersed in a portion of water for injection withstirring. In another vessel, about 10% water for injection of totalbatch size was taken and doxycycline hyclate was added with stirring todissolve. Filtered doxycycline hyclate solution was added to aboveAmberlite IRP69 dispersion and stirred for 30 minutes. The Amberlite IRP69 & doxycycline hyclate dispersion was added to the polymer mixtureobtained above with stirring and stirring was continued for 1 hr. The pHwas adjusted to 5.0 with tromethamine solution. The volume of suspensionwas finally made up to 100% batch size. The suspension was stirred for60 minutes.

Example 14

The example provides aqueous suspension formulation of Bromfenac sodium,according to one embodiment of the present invention.

TABLE 13 Bromfenac sodium suspension 0.07% w/v Example Example 15 (A) 15(B) Ingredient Function Ingredients % w/v % w/v Active IngredientBromfenac sodium 0.0805 0.0805 (equivalent to bromfenac free acid 0.07%)Anion exchange resin Indion ™860 0.07 0.07 Polymeric vehicle Carbopol974 P — 0.1 (Carbomer) Polymeric vehicle Hydroxy propyl methyl 0.3 0.3cellulose Polymeric vehicle polyvinylpyrrolidone 1.2 1.2 PreservativeBenzalkonium Chloride 0.02 0.02 Chelating agent Edetate Disodium 0.1 0.1Preservative N-Lauroylsarcosine 0.06 0.06 sodium Osmotic agent Mannitol4.5 4.5 pH adjusting agent Tromethamine q.s to qs qs adjust pH to 7.8Vehicle Water for Injection q.s. to 100 q.s. to 100

Process: In a stainless steel (SS 316) beaker, about 15% water forinjection of total batch size was taken and heated to 85° C. Thespecified polymeric vehicle, such as hydroxy propyl methyl cellulose(hypromellose 2910) was dispersed with high speed stirring to obtainuniform dispersion. The stirring was continued till temperature reached25° C. In another stainless steel (SS 316) beaker, about 12% water forinjection of total batch size was taken at 25° C. Polyvinylpyrrolidone(povidone K-90) was dispersed in water for injection with stirring toobtain uniform dispersion. In case of Example 15 (B) in a stainlesssteel (SS 316) beaker, about 10% water for injection of total batch sizewas taken and heated at 65° C. Carbopol 974P was dispersed in heatedwater for injection with stirring. The stirring was continued till thetemperature reached 25° C. The Carbopol 974P slurry was neutralized(pH7.4) with tromethamine. The hypromellose and povidone polymerdispersions obtained above were added sequentially to the carbopol 974Pphase. The polymer mixture was autoclaved at 121° C. for 20 minutes.N-lauryl sarcosine sodium was mixed in a portion of water for injectionand added to the polymer phase after filtration through 0.2 micron nylonfilter. Mannitol was dissolved in a portion of water for injection at50-60° C. and benzalkonium chloride, and edetate disodium were added toform a clear solution. This solution was added to the above polymerphase. A portion of water for injection of total batch size was taken ina vessel and Indion™860 obtained following a process similar to Example1, was dispersed with stirring. This dispersion was autoclaved at 121°C. for 20 minutes. In another vessel, a portion of water for injectionwas taken and bromfenac sodium was added with stirring to dissolve. Thissolution was filtered through 0.2 micron and 0.45 micron nylon filter.Filtered bromfenac sodium solution was added to above autoclavedIndion™860 dispersion and stirred for 30 minutes. The Indion™860 &bromfenac sodium dispersion was added to the polymer mixture obtainedabove with stirring and stirring was continued for about 30 minutes to 1hour. The volume of suspension was finally made up to 100% batch size.The suspension was stirred for about 60 minutes, followed byhomogenization at 15000 rpm for 10 mins. The pH was adjusted withtromethamine solution to about 7.8.

1-21. (canceled)
 22. A method of treating glaucoma in a patient needthereof comprising administering to the patient an ophthalmicformulation comprising an aqueous suspension containing brimonidine orits pharmaceutically acceptable salt at a concentration of from about0.05 to about 0.5% weight by volume, and a cation exchange resin,wherein the suspension is suitable for once-a-day topical instillation.23. The method of claim 22, wherein the pharmaceutically acceptable saltof brimonidine is a tartrate salt.
 24. The method of claim 22, whereinthe suspension comprises a pH adjusting agent and the pH of the aqueoussuspension is in the range of from about 7.0 to about 8.0.
 25. Themethod of claim 22, wherein the cation exchange resin is polystyrenedivinyl benzene sulphonate.
 26. The method of claim 23, wherein theweight ratio between brimonidine and cation exchange resin is in therange of from about 0.3:1 to about 1:0.3.
 27. The method of claim 22,wherein the suspension comprises one or more suspending agents selectedfrom carbopol, polyvinylpyrrolidone or hydroxypropyl methylcellulose.28. The method of claim 22, wherein a particle size distribution of thecation exchange resin particles complexed with brimonidine is such thatthe D₅₀ value is from about 50 nanometers to about 350 nanometers. 29.The method of claim 22, wherein the cation exchange resin is polystyrenedivinyl benzene sulphonate and the weight ratio between brimonidine andcation exchange resin is in the range of from about 0.3:1 to about1:0.3.
 30. A method of treating glaucoma in a patient need thereofcomprising administering to the patient an ophthalmic formulationcomprising brimonidine or a pharmaceutically acceptable salt thereof ata concentration of about 0.35% weight by volume, a cation exchangeresin, and a suspending agent, wherein the ophthalmic formulation is anaqueous suspension and is suitable for once-a-day topical instillation.31. The method of claim 30, wherein the suspending agent is selectedfrom carbopol, polyvinylpyrrolidone, hydroxypropyl methylcellulose ormixtures thereof.
 32. A method of treating glaucoma in a patient needthereof comprising administering to the patient an ophthalmicformulation comprising (a) reversible clusters of drug loadedion-exchange resin particles, said clusters have a D₅₀ value of at least2 micrometers and (b) a suspending agent, wherein the ophthalmicformulation is an aqueous suspension and the drug is brimonidine or itspharmaceutically acceptable salt at a concentration of from about 0.05%to about 0.5% weight by volume.
 33. The method of claim 32, wherein theion-exchange resin is a cation-exchange resin.
 34. The method of claim33, wherein the cation-exchange resin is polystyrene divinyl benzenesulphonate and the weight ratio between brimonidine and the cationexchange resin is from about 0.3:1 to about 1:0.3.
 35. The method ofclaim 32, wherein the suspending agent comprises one or more suspendingagents selected from carbopol, polyvinylpyrrolidone or hydroxypropylmethylcellulose.
 36. The method of claim 32, wherein the formulationcomprises a pH adjusting agent and the pH of the aqueous suspension isin the range of from about 7.0 to about 8.0.
 37. The method of claim 32,which is suitable for once-a-day topical instillation.
 38. The method ofclaim 32, wherein the drug is the tartrate salt of brimonidine.
 39. Themethod of claim 32, wherein the brimonidine or a pharmaceuticallyacceptable salt thereof is at a concentration of about 0.35% weight byvolume.
 40. The method of claim 32, wherein the drug loaded ion-exchangeresin particles deagglomerate into individual drug loaded ion-exchangeresin particles having a D₅₀ value from about 50 nanometers to about 350nanometers.